The objectives of this study are directed toward the development of an immunodiagnostic assay for the early detection of bladder cancer. A rabbit antiserum has been prepared to a Sephadex G-150 bladder cancer urine column fraction containing tumor associated antigens. After repeated absorptions with normal human serum, plasma, and concentrated (100 times) normal urine, antiserum was reacted in Ouchterlony gel diffusion assays against urine samples concentrated 100 times. None of the normal urines showed positive bands. 62.5% of the papillomas, 60% of in situ tumors, 87.5% grade I tumors, 90% grade II tumors, 100% grade III tumors and 100% grade IV tumors were positive. Also, 37% of non-cancer but clearly infected urines were positive. This antiserum was then reacted in a microcomplement fixation (CF) system. To eliminate anticomplementary activity the antiserum was absorbed utilizing a glutaraldehyde matrix consisting of normal urine and plasma. All normal samples (n equals 25) had mean 50% fixation values greater than 120 micrograms Mean 50% CF values were 35 micrograms plus or minus 10 for papilloma urine, 40 micrograms plus or minus 15 for in situ urines, 30 micrograms plus or minus 10 for grade I urines, 15 micrograms plus or minus 5 for grade II urines, 20 micrograms plus or minus 12 for grade III urine, 5 micrograms for grade IV urines and 115 plus or minus 25 for infected urines. Thus, these data suggest that quantitative antigenic differences may exist between infected and cancer may be important in the early diagnosis of bladder cancer.